With the aim of realizing simple quantification of body fluid components by ordinary people, various types of biosensors have recently been developed which utilize the specific catalytic action of enzymes.
The following will describe a method of glucose quantification as one example of the method of quantifying a component contained in a sample solution. As an electrochemical method of glucose quantification, a method using the combination of glucose oxidase and an oxygen electrode or a hydrogen peroxide electrode is generally well-known (see “Biosensor” ed. by Shuichi Suzuki, Kodansha, for example).
Glucose oxidase selectively oxidizes glucose as a substrate to gluconolactone using oxygen as an electron mediator. In the process of the oxidation reaction of glucose by glucose oxidase in the presence of oxygen, oxygen is reduced to hydrogen peroxide. The decreased amount of oxygen is measured by the oxygen electrode, or the increased amount of hydrogen peroxide is measured by the hydrogen peroxide electrode. Since the decreased amount of oxygen and the increased amount of hydrogen peroxide are proportional to the content of glucose in the sample solution, glucose quantification is possible based on the decreased amount of oxygen or the increased amount of hydrogen peroxide.
As speculated from the reaction process, the above method has a drawback in that the measurement is affected by the oxygen concentration of the sample solution, and if the oxygen is absent in the sample solution, the measurement is infeasible.
Under such circumstances, glucose sensors have been developed which use, as the electron mediator, an organic compound or a metal complex such as potassium ferricyanide, a ferrocene derivative or a quinone derivative without using oxygen as the electron mediator. In the sensors of this type, the reduced form of the electron mediator which results from the enzyme reaction is oxidized on an electrode, and based on the amount of this oxidation current, the concentration of glucose contained in the sample solution can be determined. With the use of such an organic compound or metal complex as the electron mediator in place of oxygen, it is possible to form a reagent layer in which the electron mediator is carried with glucose oxidase on the electrode in a precise amount and in a stable state. Further, it is also possible to integrate the reagent layer, in an almost dry state, with the electrode system, and hence disposable glucose sensors based on this technique have recently been receiving a lot of attention. A typical example thereof is a biosensor disclosed in Japanese Patent Publication No. 2517153. With such a disposable glucose sensor, by simply introducing a sample solution into the sensor connected detachably to a measurement device, glucose concentration can be measured readily by the measurement device.
According to the measurement method using the above-described glucose sensor, the concentration of glucose in a sample can be determined in about 30 seconds based on the response current which is in the order of 1 to 10 μA/cm2. However, in recent years, it is anxiously desired in various fields to develop sensors capable of quantifying glucose more rapidly and with higher sensitivity.
Also, in conventional electrochemical glucose sensors, by the addition of a hydrophilic polymer such as carboxymethyl cellulose to the reagent layer, the measurement is prevented from being affected by vibrations given to the measurement device from outside. The hydrophilic polymer has another merit that it functions as a binder to immobilize the enzyme on the electrode moderately. The presence of the hydrophilic polymer, however, may cause changes in catalytic activity of glucose oxidase or thermodynamics of the hydrolytic reaction from gluconolactone to gluconic acid, thereby to cause accumulation of gluconolactone, which is a product of the oxidation reaction of glucose. The accumulation of gluconolactone causes the reverse reaction to proceed and causes the rate of the oxidation reaction of glucose to decrease. As a result, the amount of the reduced form electron mediator generated in a short reaction time is reduced, thereby resulting in a decrease in the magnitude of the response current to glucose, i.e., the sensitivity of the sensor. Particularly, in order to obtain a sufficient sensitivity to high concentrations of glucose, it becomes necessary to increase the reaction time for generating large amounts of the reduced form electron mediator, so that the measurement tends to require longer time.
In view of the above conventional problems, an object of the present invention is to provide a biosensor that is highly responsive and capable of rapid and highly sensitive quantification of a specific component in a sample.